Acetycholine released from nerve terminals interacts with cholinergic receptors to initiate cellular responses. Two major class of cholinergic receptors (nicotinic and muscarinic) have been defined by their pharmacological specificity and anatomical location. On the basis of the pharmacological characterization of various in vivo and in vitro responses, as well as non-classical kinetics observed in studies of radioligand binding assays, it has been suggested that subtypes of muscarinic cholinergic receptors exist. These studies, however, have yielded equivocal results. The goals of this proposal are to examine the hpothesis that subtypes of muscarinic receptors exist and, if they do exist, to examine the in vivo regulation of these receptor subtypes. Two independent methods are proposed to examine the question of the existence of subtypes. First, using the antagonist 3H-quinuclidinyl benzilate to label muscarinic receptors, the pharmacological properties of the inhibition of the binding of this radioligand by a variety of drugs will be examined in the presence and absence of guanine nucleotides. This should eliminate the contribution of ternary complex (ligand.receptor.guanine nucleotide binding protein) formation to the binding of drugs to muscarinic recepotrs. A variety of tissues will be examined to attempt to identify several which contain only one or at least a preponderance of one subtype. Clonal cells grown in tissue culture may help in this regard. The shape of the inhibition curves of many drugs will be computer analyzed to determine the nunber and properties of the subtypes present in a given tissue. The second approach will involve the pharmacological characterization of two biochemical responses associated with activation of muscarinic cholinergic receptors: 1) Stimulation of phosphatidylinositol turnover and 2) Inhibition of adenylate cyclase activity or cyclic AMP accumulation. Analogy with results obtained with Alpha-adrenergic, histaminergic and serotonergic receptors, suggests that the former function is mediated by a different receptor subtype than is the latter function. Careful characterization of these responses in a number of different tissues including cultured cells, may enable us to subclassify muscarinic receptors on the basis of response. A comparison of the pharmacological profiles of the binding and the biochemical responses should yield unequivocal evidence that subtypes of muscarinic cholinergic receptors exist. Finally, the regulation of these putative subtypes and their biochemical responses by alterations in the availability of acetylcholine will be examined. These studies should yield information as to the anotomical location of each subtype and the ability of each subtype to respond to changes in their functional environment.